The enzyme lecithin-cholesterol acyltransferase (LCAT) circulates in the plasma portion of blood in association with high-density lipoproteins (HDL). LCAT catalyzes the hydrolysis of fatty acids from phosphatidylcholine (PC) within HDL and subsequently transfers most of that fatty acid to an ester bond with cholesterol within the same HDL to form cholesteryl ester (FIG. 1). A smaller portion of the fatty acid removed from PC may be released to water as free fatty acid. Of the two fatty acyl chains in PC, the fatty acid at the sn-2 position of the glycerol backbone in PC is the principle target of LCAT although the fatty acid esterified at the sn-1 position can also be hydrolyzed.
Apolipoprotein AI (apoA-I), which is the major protein component of HDL, is an essential cofactor for activation of LCAT. A matrix of PC and cholesterol alone, without apoA-I or similar activator, reacts slowly with LCAT. Other LCAT activity-enhancers, in addition to apoA-I, are known. These include the apolipoproteins E, A-IV and C-I. Synthetic peptides capable of activating LCAT in the same manner as apoA-I have also been described.
Cholesterol is the natural and, likely, most active substrate for LCAT but other sterols are known to be substrates for esterification by LCAT. The other sterols include phytosterols, steroid hormones such as estradiol, and fungal sterols.
The enzymatic activity of LCAT in plasma is essential for maintaining good health. Persons with the genetic disease familial LCAT deficiency (FLD) lack functional LCAT in their blood and, as a result, develop corneal opacities, anemia, and kidney disease which inevitably leads to kidney failure before the fourth decade of their life. A milder form of LCAT deficiency also occurs and is known as fish eye disease (FED) in which corneal opacity is the only clinical symptom. There is also evidence of an increased risk of vascular disease in FED. Both FLD and FED result from mutations in the LCAT gene.
Diminished plasma LCAT activity is also associated with increased illness in persons with a normal LCAT gene. For example, plasma LCAT activity is lower than normal in persons with chronic kidney disease, in those suffering cardiovascular disease, in liver disease, during sepsis, and in rheumatic disease patients.
Understanding and diagnosing the links between health and proper LCAT function as well as assessing the benefit of current therapies and the development of new therapies is dependent on reliable and facile methods for measuring LCAT activity.
A variety of assay protocols have been described for measurement of LCAT activity. They vary, in part, according to the particular LCAT enzymatic chemistry to be assayed, since there are two principle reactions catalyzed by LCAT. Cholesterol esterification by transacylation of fatty acid from phosphatidylcholine to cholesterol to form cholesteryl ester is the principal activity of LCAT and, correspondingly, the most broadly assayed. Many assay protocols are described and a commercial kit is available for measuring cholesterol esterification Water can also serve as an acyl-chain acceptor in the transacylation reaction, in which case phospholipid hydrolysis is the enzymatic activity measured. Assay procedures and kits have been described that measure the hydrolytic deacylation of phospholipids by LCAT. A third, but far less common type of LCAT assay, measures the LCAT-dependent transacylation of a fatty acid moiety from phosphatidylcholine to lysophosphatidylcholine.
Most cholesterol esterification assays use radiolabeled cholesterol incorporated into an appropriate substrate matrix, typically a phosphatidylcholine and cholesterol mixture complexed with apoA-I, and measure the appearance of radioactive cholesteryl ester due to LCAT. The substrate complex is usually formed by combination of the individual substrate components but the use of radiolabeled cholesterol incorporated into natural plasma lipoproteins in plasma or serum has also been described. Some have described procedures to enhance the detection of LCAT activity with plasma HDL, the target substrate of LCAT, by first removing non-HDL lipoproteins from plasma or serum before the addition of radiolabeled cholesterol and subsequent measurement of radiolabeled cholesteryl ester formation.
In lieu of radioactivity, some cholesterol esterification assays are based on the direct measurement of cholesterol depletion due to esterification for example, by cholesterol mass detection techniques (e.g. gas chromatography) or enzyme-based colorimetric assays of cholesterol.
Some assay methods for detecting LCAT activity are designed to detect the deacylation of phosphatidylcholine alone, in the absence of cholesterol. These assay methods either use radiolabeled phosphatidylcholine substrate in which the appearance of radiolabeled fatty acid or lysophosphatidylcholine is detected, or they use a phosphatidylcholine analog containing a fluorescent moiety that can be monitored for changes in spectral properties as a result of phosphatidylcholine deacylation by LCAT.
Two types of kits for the assay of LCAT are available for purchase from vendors, but none functions in the manner of the assay presented herein. One of the available kits is based on detection of phosphatidylcholine hydrolysis by monitoring fluorescence changes in a fluorescent PC analog as a result of LCAT activity (Roar Biomedical, Inc., New York, N.Y.). The second type of kit uses an enzyme-based colorimetric assay of cholesterol to detect the decline in free cholesterol in a cholesterol:phosphatidylcholine complex as a result of cholesterol esterification by LCAT (Sekisui Medical Co., Ltd., Tokyo, Japan).
The primary and, thus, most relevant activity of LCAT to detect and measure by assay is the transacylation of fatty acid from PC to cholesterol to form cholesteryl ester. FIG. 1 depicts the LCAT reaction in which LCAT catalyzes the transacylation of the fatty acyl group from the sn-2 position in the glycerol backbone of phosphatidylcholine to cholesterol. The products of the reaction are lysophosphatidylcholine and cholesteryl ester.
The most sensitive and most widely used LCAT assay procedures are based on the detection of radiolabeled cholesteryl ester formation from radiolabeled substrates. While the radiolabel-based assays are popular they are also cumbersome and lengthy due to the need to employ lipid extraction and chromatography steps to enable resolution of radiolabeled substrate and product for quantitation.
Measurement of LCAT activity is greatly simplified when, rather than analyzing radiolabeled products, enzymatic assay of unesterified cholesterol before and after reaction with LCAT is used to deduce the change in free cholesterol which relates directly to the amount of CE product formed. The enzymatic assay of cholesterol is much more efficient since extraction and separation of cholesterol and CE product is not necessary. Despite this advantage over the radiolabel method, the shortcoming of the enzymatic method is that it is much less sensitive and, thus, prone to greater error in detection of low levels of LCAT or of small changes in LCAT activity. There is a need for an LCAT assay procedure that overcomes these various shortcomings.